Volume 67, Issue 2, Pages 213-223 (July 2010) New Neurons Clear the Path of Astrocytic Processes for Their Rapid Migration in the Adult Brain  Naoko Kaneko, Oscar Marín, Masato Koike, Yuki Hirota, Yasuo Uchiyama, Jane Y. Wu, Qiang Lu, Marc Tessier-Lavigne, Arturo Alvarez- Buylla, Hideyuki Okano, John L.R. Rubenstein, Kazunobu Sawamoto  Neuron  Volume 67, Issue 2, Pages 213-223 (July 2010) DOI: 10.1016/j.neuron.2010.06.018 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 New Neuron Migration in the RMS of S1−/− Mice (A and B) Hoechst-stained sagittal brain sections including the RMS of P7 WT (A) and S1−/− (B) mice. In the S1−/− brain, cells accumulated within the anterior part of the SVZ and proximal part of the RMS (arrows), whereas the distal part of the RMS and its extension inside the OB (arrowheads) were thinned. (C–E) Time-lapse sequence of DiI-labeled cells migrating in the RMS of WT (C) and S1−/− (D) brain slices (OB is to left). Four cells in each slice are labeled (1, blue; 2, light green; 3, red; 4, cyan) in the 0 min panel, and their tracks over time are indicated by lines of the same color. The mean migration speed of DiI-labeled cells in the S1−/− RMS (yellow bar) was significantly slower than in the WT RMS (gray bar) (E, p < 0.0001). (F–K) AP staining of WT (F) and S1−/− (G) brains 3 days after AP-encoding retroviral injection into the anterior SVZ (J). Arrows in (F) and (G) indicate AP-labeled migrating new neurons in the SVZ-RMS-OB pathway (between broken lines). Black lines indicate the boundaries of the SVZ, RMS, and OB. (H) and (I) are higher-magnification images of the boxes in (F) and (G), respectively. The percentage of AP-labeled cells inside the OB was significantly lower in the S1−/− than the WT brain (K, p = 0.0011). Error bars indicate ±SEM. Scale bars: 2 mm (A and B), 1 mm (F and G), 500 μm (H and I), 200 μm (C and D). See also Movie S1. Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Migration of WT and S1−/− Cells Transplanted into WT and S1−/− Brain Slices (A) Schematic drawing of the experimental protocol. New neurons dissociated from WT and S1−/− SVZs were labeled with different fluorescent dyes, red (PKH-26) for WT and green (PKH-67) for S1−/−, mixed into cell aggregates, and transplanted into the anterior SVZ of WT and S1−/− sagittal brain slices. The migration of the labeled cells was recorded by two-color time-lapse imaging. (B–E) PKH-labeled transplanted WT (red) and S1−/− (green) cells migrating in a WT (B) or S1−/− (D) brain slice. Scale bars indicate 200 μm. OB is to the left. The cell tracks during 4 hr of monitoring are shown by the colored lines in (C) (WT) and (E) (S1−/−) (closed circles, cell positions at 0 min; open circles, positions at 240 min). (F) Mean speed of migration of WT and S1−/− new neurons transplanted into WT (gray bar, WT cells; dark green bar, S1−/− cells) or S1−/− RMS (orange bar, WT cells; yellow bar, S1−/− cells). Compared with the speed of WT cells transplanted into WT slices, the speed of S1−/− cells transplanted into WT slices (p < 0.0001) and WT cells transplanted into S1−/− slices (p = 0.0002) was significantly reduced. The S1−/− cell migration speed was even slower in S1−/− slices (S1−/− cells in WT RMS versus S1−/− cells in S1−/− RMS: p = 0.0041, WT cells in S1−/− RMS versus S1−/− cells in S1−/− RMS: p = 0.0007). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Error bars indicate ±SEM. See also Movie S2. Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Localization of Robo2 Protein in the RMS (A) Slit1/GFP and Robo2 immunohistochemistry in the RMS. Slit1/GFP (green) was localized to chain-forming new neurons in the RMS. Robo2 (red) protein was also detectable along the RMS, including in new neurons. Note that the Robo2+ processes with astrocytic morphology surrounding the chains of new neurons were negative for Slit1 (arrowheads). The nuclei were stained with TOTO3 (blue). (B and C) Robo2 immunohistochemistry in the RMS. Robo2 protein (green) was detectable all along the RMS (B). High-magnification images. Boxed area in (B) shows that Robo2 (green) was strongly detected in the GFAP+ (red) astrocytic soma and processes and weakly in the Dcx+ (blue) new neurons (C). (D and E) Robo3 immunohistochemistry in the RMS (E) is a high-magnification image of the boxed area in (D). Robo3 protein (green) was strongly detected in neurons in the neocortex and striatum (open arrowheads in E) and more weakly in the RMS. Within the RMS, the immunoreactivity of astrocytic processes (red) was easily detected, whereas that of new neurons (blue) was faint. (F and G) Immunocytochemistry of cultured SVZ/RMS cells dissociated from adult WT mice. Robo2 (green) was localized to the GFAP+ (red) processes (arrows) and the soma of astrocytes, and to Dcx+ (blue) new neurons (arrowhead) (F). High-magnification images of the boxed area in (F) are shown in (G). Scale bars: 200 μm (B and D), 50 μm (C, E, and F), 20 μm (A and G). Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Cellular Organization of the RMS in S1−/− Mice (A–E) Confocal images of the RMS of WT (A and B) and S1−/− (C and D) mice stained with Dcx (red) and GFAP (green). Sagittal sections of the WT RMS showed GFAP+ (green) astrocytic processes parallel to Dcx+ (red) new-neuron chains (A). The S1−/− RMS showed more, thick GFAP+ processes with irregular orientations, occasionally running across the chains (C). Coronal slices of WT RMS (B) showed cross-sections of thin GFAP+ astrocytic processes between clusters of Dcx+ neurons. The S1−/− RMS (D) showed many longitudinal sections of thick GFAP+ processes among the new neurons. Quantification of the GFAP+ area in the RMS showed that Slit1 deletion caused a significant increase in the amount of astrocytic processes within the RMS (E, p = 0.0012). (F–K) Ultrastructural organization of the WT (F and G) and S1−/− (H and I) RMS, according to the criteria described in Experimental Procedures. New neurons (dark cytoplasm) and astrocytes (light cytoplasm) are indicated by red and blue, respectively (G and I). The percentage of astrocyte cell bodies in the RMS was not significantly different between the two groups (J, p = 0.9558); however, the astrocytic processes within chains were significantly more frequent in the S1−/− RMS (I, arrows) compared to controls (G). The area occupied by astrocytic processes inside the chains of new neurons was significantly increased in the S1−/− RMS (K, p = 0.0409). Error bars indicate ±SEM. Scale bars: 50 μm (A and C), 20 μm (B and D), 5 μm (F–I). See also Figure S1. Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Repulsive Activity of Slit for Cultured Astrocytes (A-D) SVZ/RMS astrocytes were cocultured with Slit-secreting (pink) and control (pale green) HEK cells mixed with collagen gel (A). Boxes in (A) indicate the magnified area shown in (B) and (C). After 10 hr, most astrocytes were attached to the dish and had short processes; few were on gel pieces containing control (B) or Slit-expressing (C) HEK cells. After 4 days, several astrocytes had migrated onto the pieces containing control HEK cells (B–B′, arrows), but significantly fewer onto those containing Slit-secreting (C–C′) HEK cells. The proportion of the 500 μm wide strip just inside the edge of the gel piece that was GFAP+ was quantified (D, p = 0.0094). (E–K) Effects of Slit on astrocytes transfected with dominant-negative Robo (Robo DN). Cultured astrocytes dissociated from the SVZ and RMS were transfected with GFP-tagged Robo DN (H and I) or GFP (F and G), then cocultured with Slit-secreting or control HEK cells in collagen gel for 60 hr (E). Images show GFP+ cells near the edge of the control (F and H) and Slit-containing (G and I) HEK-cell-mixed gel. Graph shows the percentage of GFP+GFAP+ astrocytes on the gel within 500 μm of its edge divided by the number in the 1000 μm wide space from 500 μm inside to 500 μm outside the gel border (J and K). The percentage of GFP-transfected astrocytes was significantly lower on Slit-containing gel pieces than on controls (J, p = 0.0026). This difference was not seen using astrocytes transfected with Robo DN-GFP (K). Error bars indicate ±SEM. Scale bars: 200 μm. See also Figure S2. Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Slit1-Robo-Dependent Migration of New Neurons on Astrocytes Migration speed of new neurons on an astrocytic feeder layer. Time-lapse sequence of WT (A) and S1−/− (B) new neurons migrating on the surface of plastic dishes without astrocytes. Representative tracks of the migrating new neurons indicated by dots and lines showed no significant difference in the migration speed between the two groups (E, p = 0.2850). Time-lapse sequence of WT (C) and S1−/− (D) new neurons migrating on astrocytes dissociated from the SVZ/RMS of Gfap-EGFP mice. The tracks of representative cells indicated by dots in the first panels are shown by lines in the following panels (16 min, 32 min, 48 min, 64 min). The migration speed of S1−/− new neurons on astrocytes was significantly reduced compared with that of WT new neurons (F, p = 0.0012). Error bars indicate ±SEM. Scale bars: 50 μm. See also Figure S3. Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Dynamic Morphological Changes of Astrocytes Associated with Neuronal Migration (A–C) New neurons (Dcx-DsRed) and SVZ/RMS astrocytes (Gfap-EGFP) were cocultured in 3D matrices (A). New neurons (red) migrated along the processes of astrocytes (green), on which furrows occasionally formed (B, arrows). A higher-magnification image of the boxed area in (A) is shown in (C). At right, a series of X-Z sections of the furrow on an astrocyte at the positions indicated by the green lines shown in the panel at left. (D and E) Time-lapse recording of furrow formation. Middle and right panels show images captured 10 and 30 min after the left one. When new neurons migrated on a monolayer culture of SVZ/RMS astrocytes transfected with a control vector encoding GFP (D), the astrocytes changed their shape, occasionally forming furrows on the membrane in contact with the new neurons (arrows). Such morphological changes were suppressed in astrocytes transfected with dominant-negative Robo1 (Robo DN) (E). (F) Quantification of the furrows. Graph shows the number of furrows formed on 1 mm2 of the astrocyte membrane surface per hour, which was significantly reduced for astrocytes transfected with Robo DN compared with control-vector-transfected astrocytes (p = 0.0254). (G and H) Higher-magnification images of the boxed regions in (D) and (E), respectively. The migration of new neurons (asterisks) over astrocytes was frequently associated with the formation of furrows on the membrane of astrocytes transfected with the control vector (G) but not with Robo DN (H). (I) The migration speed of new neurons on Robo-DN-transfected astrocytes was significantly decreased compared with that on GFP-transfected astrocytes (p < 0.0001). Error bars indicate ±SEM. Scale bars: 20 μm. See also Figure S4 and Movies S3, S4, and S5. Neuron 2010 67, 213-223DOI: (10.1016/j.neuron.2010.06.018) Copyright © 2010 Elsevier Inc. Terms and Conditions